The present invention concerns a biological material comprising a matrix consisting of at least one derivative of hyaluronic acid on which endothelial cells, glandular cells such as islets of Langerhans and liver cells, skin adnexa and germinative cells of hair bulbs are grown.
It is possible nowadays to reproduce angiogenesis, that is, the formation of new blood vessels, experimentally in vitro by various means and using different stimulants such as Vascular Endothelial Growth Factor (VEGF) or basic Fibroblast Growth Factor (bFGF) (R. Montesano et al., PNAS USA, 1986, 83; 7297-7301, xe2x80x9cBasic Fibroblast Growth Factor induces angiogenesis in vitroxe2x80x9d; J. Folkman et al., PNAS USA, 1979, 76; 5217-5221, xe2x80x9cLong term culture of capillary endothelial cellsxe2x80x9d; R. Montesano et al., Lab. Invest., 1996, 75; 249-262, xe2x80x9cSynergistic effect of hyaluronan oligosaccharides and vascular endothelial growth factor on angiogenesis in vitroxe2x80x9d).
The reorganization of endothelial cells into tubular structures has been observed, for example, in the presence of collagen in the course of gelation, or between double layers of collagen.
Even more encouraging results have recently been obtained using as a culture support basement membrane extracts (Matrigel), on which the angiogenic mechanism seems more rapid and more easily reproducible. It was thus possible to demonstrate that the presence of a scaffold containing collagen fibers facilitates cell differentiation which, in the case of endothelial cells, translates into the organization of a thin web of tubular structures similar to that found in the extracellular matrix of connective tissues (J. Folkman et al., Nature, 1980, 288, 551-556, Angiogenesis in vitroxe2x80x9d; J. A. Madri et al., J. of Cell Biol., 1983, 97, 153165, xe2x80x9cCapillary endothelial cell culture: phenotypic modulation by matrix componentsxe2x80x9d).
It is well known that matrices of partial or total esters of hyaluronic acid with benzyl alcohol (HYAFF(copyright)) in the form of nonwoven fabric are suitable for the in vitro growth and development of various cell types such as fibroblasts (WO 96/33750).
WO 97/18842 refers to a culture of autologous or homologous bone marrow stem cells partially or completely differentiated into cellular lines of a specific connective tissue and the extracellular matrix produced by said connective tissue, said cells growth onto a scaffold of a three-dimensional biocompatible and biodegradable matrix consisting of a hyaluronic acid derivative. The success of this biological material was because the cells used are very active and can be suitably differentiated into various cell lines when placed onto the matrix. From these stem cells it is possible to obtain differentiated cells such as fibroblasts, adipocytes, myoblasts, osteoblasts and chondrocytes.
Weak and fragile differentiated cells such as endothelial, glandular cells, islets of Langerhans, liver cells or skin adnexa are more difficult to be isolated and cultured onto artificial or plastic support than staminal cells and they show poor proliferative properties and short survival times.
For example, liver cells can survive in vitro for about 7 weeks with less than 50% of the cells remaining viable (J. C. Gerlach et al., Hepatology August 1995, Vol. 22 No. 2, pages 546-552), while skin adnexa last about two weeks (A. Limat et al., The Journal of Investigative Dermatology, Vol. 87, No. 4 October 1986, pages 485-488), and islets of Langerhans just a few days (S. G. Matta, Pancreas, Vol. 9, No. 4, 1994, pages 439449).
It therefore follows that although the properties of HYAFF(copyright) matrices are already known to favour the growth and development in vitro of resistant and very active cellular elements such as staminal cells or fibroblasts, ecc. an expert in the field would have not be able to predict that satisfactory proliferation rates and survival times can be achieved by cultivating cell types like poor resistant, weak and with short time of survival cells as above said.
The authors of the present invention have instead surprisingly found that also poor resistant and weak cells such as endothelial cells, glandular cells and skin adnexa, germinative cells of hair bulbs, ecc. can efficiently grow on a hyaluronic acid derivative matrix.
A characteristic of the present invention is, therefore, a biological material comprising:
a) at least one cell type selected from the group consisting of endothelial cells, glandular cells, skin adnexa, germinative cells of hair bulbs and optionally keratinocytes; and
b) a biocompatible and biodegradable three-dimensional matrix comprising at least one hyaluronic acid derivative and optionally collagen and/or fibrin.
The authors of the present invention have furtherly surprisingly found that when the above said cells, comprised in the biological material according to the inventions are cultivated in particular culture conditions such as in presence of a medium treated with fibroblasts or in a co-culture with fibroblasts seeded on the biomaterial, at different times, preferably several days, previously or at the same time as the cells, the proliferation rate is significantly higher than that which is achieved using other supports in the same conditions.
A further aspect of the present invention is therefore a biological material comprising:
a) at least one cell type selected from the group consisting of endothelial cells, glandular cells, skin adnexa, germinative cells of hair bulbs, and optionally keratinocytes cultured in presence of a medium treated with fibroblasts or in a co-culture with fibroblasts; and
b) a biocompatible and biodegradable three-dimensional matrix comprising at least one hyaluronic acid derivative and optionally collagen and/or fibrin.
Another aspect of the present invention is a process for the preparation of a biological material according to the invention.
The invention relates also to the use of the biological material according to the invention for human and veterinary use, in cardiovascolar and oncological surgery, in transplants, to enhance the biological process of tissue vascularization and for aesthetic use.
Furtherly, another aspect is the use of the biological material according to the invention for the screening of medicaments or toxic substances and as a support for gene transfection.